Kidney-specific urate transporter and gene thereof

ABSTRACT

It is intended to identify and provide a novel urate transporter gene participating in the urate transport in the kidney and a urate transporter which is a polypeptide encoded by the above gene. Namely, a protein comprising the amino acid sequence represented by SEQ ID NO:1 or an amino acid sequence derived therefrom by deletion, substitution or addition of one to several amino acids and being capable of transporting uric acid and its analogs; and a gene encoding this protein.

TECHNICAL FIELD

[0001] The present invention relates to a gene participating intransport of uric acid and analogs thereof or exchange transport of uricacid and the other anion, and a polypeptide encoded by the gene.

BACKGROUND ART

[0002] In human race and primates, uric acid which is an organic acid isa final metabolite in purine metabolism in cells, and is excreted mainlyfrom the kidney. In species other than the human race and the primates,it is metabolized to allantoin by an action of uricase in liver, and isexcreted from the kidney. Therefore, for the other mammals, it seemsthat effects of dynamic abnormality of uric acid which is anintermediate product in the kidney on living body are small. Losing theaction of uricase in the evolution process seems to be a cause of thefact that the human race has suffered from gout due to hyperuricemiasince ancient times.

[0003] In humans, when is caused the decrease of uric acid excretion inthe kidney causes hyperuricemia, the gout develops at high percentage,which becomes a risk factor for cardiovascular diseases andhypertension. On the other hand, it has been known that the increase ofuric acid excretion in the kidney causes renal hypouricemia. Althoughabnormality of uric acid kinetics is not obvious in these diseases, ithas been supposed that urate transporters in the kidney are deeplyinvolved.

[0004] The uric acid kinetics in the kidney has been studied byexperimental systems using a removed organ perfusion method and anisolated cell membrane vesicle system. In humans, it has beendemonstrated that uric acid freely passes through renal glomerulus andthereafter mechanisms for reabsorption and secretion exist in proximalconvoluted tubule. However, by the conventional technique, it has beendifficult that urate transport system via cell membrane is analyzed indetail, and it has been desired that the transporter per se is isolatedand analyzed.

[0005] It has been known that there is a remarkable difference amongspecies in the urate transport in the kidney, and there exist thespecies where secretion is dominant such as swine and rabbit and thespecies where the reabsorption is dominant such as human, rat and dog.The swine of the species with secretion dominance excretes from 200 to300% of uric acid per unit nephron, whereas a human of the species withuric acid reabsorption dominance excretes only about 10% of uric acidper unit nephron. Also, it has been known that responses to uricosuricaccelerators and uricosuric inhibitors are different even among thespecies with reabsorption dominance. Accordingly, since the kinetics ofuric acid and the responses to drugs in the kidney are differentdepending on the species, and uric acid is reciprocally transported, ithas not been easy to isolate a molecular entity of the urate transporterthough its existence has been assumed.

[0006] Among the urate transporters in the kidney, the transporterswhich reabsorb uric acid from renal tubular lumen have been studied forlong time by the experimental system using the isolated cell membranevesicle system. For the drugs currently used for the patients withhyperuricemia and gout, it is assumed that the transporter whichreabsorbs uric acid in the kidney is inhibited. Also, it is forecastedthat renal hypouricemia is caused due to gene aberration of thistransporter.

[0007] Recently, it has been demonstrated that the transporters involvedin the reabsorption of uric acid are exchange transporters of uric acidand various anions in several experiments. For pyrazinamide used as thefirst-line drug of antituberculous drugs at present, it has been shownthat pyrazine carboxylate which is the metabolite of pyrazinamide is anexchange substrate of this exchange transporter and facilitates thereabsorption of uric acid. That is thought to be the cause ofhyperuricemia frequently observed in the patients administered theantituberculous drug.

[0008] Accordingly, the transporter involved in the reabsorption of uricacid in the kidney is thought to play an important role for internalkinetics of uric acid. It has been anticipated that elucidation of itsmolecular entity leads to elucidate a mechanism of action of uricosuricaccelerators and a cause of renal hypouricemia, and development of newgout curative medicines.

[0009] We have previously isolated and reported organic aniontransporters, OAT1 (organic anion transporter) (Sekine, T. et al., J.Biol. Chem., 272:18526-18529, 1997), OAT2 (Sekine, T. et al., FEBSLetter, 429:179-182, 1998), OAT3 (Kusuhara, H. et al., J. Biol. Chem.,274:13675-13680, 1999), and OAT4 (Cha, S. H. et al., J. Biol. Chem.,275:4507-4512, 2000) which play central roles in medicament transport inthe kidney, liver, brain, placenta and so on. These transportersbelonging to OAT family are the transporters capable of transportingmany organic anions with different chemical structures, and also performthe transport of various anionic medicaments.

[0010] It was not obvious whether the urate transporter belongs to theknown transporter family, but since uric acid is a dibasic acid havingboth pyrimidine structure and imidazole structure and is one of theorganic anions, the possibility that the urate transporterphylogenetically belonges to OAT family was anticipated. In OAT family,since OAT4 exists at the side of renal tubular lumen in the kidney andthe existence of the transporter involved in the reabsorption of uricacid is also assumed at the side of renal tubular lumen, it has beenalso anticipated that the transporter is phylogenetically similar toOAT4.

[0011] From these facts, we have anticipated that the urate transporterin the kidney belongs to the organic ion transporter family.

DISCLOSURE OF THE INVENTION

[0012] An object of the present invention is to identify and provide anovel urate transporter gene participating in the urate transport in thekidney and a urate transporter which is a polypeptide encoded by theabove gene. Other objects will be apparent from the followingdescription.

BRIEF DESCRIPTION OF THE DRAWINGS

[0013]FIG. 1 shows the results of analyzing the expression of URAT1 genemessenger RNA in various organ tissues of human adult and embryo byNorthern blotting.

[0014]FIG. 2 shows the result of time dependency in uric acid uptakeexperiments by oocytes injected with cRNA of URAT1 gene.

[0015]FIG. 3 shows the result of concentration dependency in uric aciduptake experiments by oocytes injected with cRNA of URAT1 gene.

[0016]FIG. 4 shows the result of examining the effects of added salts inuric acid uptake experiments by oocytes injected with cRNA of URAT1gene.

[0017]FIG. 5 shows the result of pH dependency in uric acid uptakeexperiments by oocytes injected with cRNA of URAT1 gene.

[0018]FIG. 6 shows the result of preincubation with various organicacids in uric acid uptake experiments by oocytes injected with cRNA ofURAT1 gene.

[0019]FIG. 7 shows the result of examining the effect of previouslyinjected unlabeled lactic acid (100 mM, 10 nl) in uric acid uptakeexperiments by oocytes injected with cRNA of URAT1 gene.

[0020]FIG. 8 shows the result of examining the effects of addition ofvarious organic acids or analog compounds thereof to the system in uricacid uptake experiments by oocytes injected with cRNA of URAT1 gene.

[0021]FIG. 9 shows the result of examining the effects of probenecidaddition at various concentrations to the system in uric acid uptakeexperiments by oocytes injected with cRNA of URAT1 gene.

[0022]FIG. 10 shows the result of examining the effects of losartanaddition at various concentrations to the system in uric acid uptakeexperiments by oocytes injected with cRNA of URAT1 gene.

[0023]FIG. 11 shows exon-intron structure of URAT1 gene in human genome.

BEST MODE FOR CARRYING OUT THE INVENTION

[0024] As described above, the present inventors isolated four organicanion transporters, OTA1, OTA2, OTA3 and OTA4. They have about 40%homology of amino acid sequences each other. On the basis of thesesequences, disclosed information of human genome project was searched,and multiple novel gene fragments having homology to OAT1, 2, 3 and 4were identified. Among them, one novel gene fragment extremely closed toa gene locus position of OAT4 was analyzed, and a site supposed to be aninitiation codon was identified. A primer specific for 5′ upstream ofthis initiation codon was made, and isolation of this novel gene wasattempted by 3′-RACE (3-rapid amplification of cDNA ends) method usingmessenger RNA derived from various tissues of humans. As a result, anovel clone (URAT1) which had been never reported was identified by the3′-RACE method using human kidney messenger RNA.

[0025] The urate transporter1, URAT1 of the present invention has anability to transport uric acid and its analogs via cell membrane fromone side to the other side and further is a urate/anion exchanger bymaking the anion at the other side of the cell membrane an exchangesubstrate.

[0026] The protein of the present invention includes, for example, thosehaving the amino acid sequence in which one or several amino acids aredeleted, substituted or added in the amino acid sequence represented bySEQ ID NO:1, in addition to one having the amino acid sequencerepresented by SEQ ID NO:1. The amino acids could be deleted,substituted or added to the extent where urate transport activity is notlost, and typically from 1 to about 110 and preferably from 1 to about55. Such proteins typically have up to 75% and preferably up to 90%homologous amino acid sequences to the amino acid sequence representedby SEQ ID NO:1.

[0027] In the present invention, the isolation of the gene by the3′-RACE method can be carried out typically by making a primer of about30 bases specific for guanine- or cytosine-rich gene at the 5′ upstreamof the initiation codon, performing reverse transcription oftissue-derived messenger RNA using an oligo dT primer with an adaptersequence, and subsequently performing PCR (polymerase chain reaction)using the adapter sequence and the gene-specific primer. It is possibleto further enhance accuracy of the PCR by the use of heat resistantpolymerase with higher fidelity.

[0028] The urate transporter gene of the present invention can beisolated and yielded by screening cDNA library prepared using renaltissues or cells in an appropriate mammal as a gene source. The mammalsinclude human in addition to non-human animals such as dog, cattle,horse, goat, sheep, monkey, swine, rabbit, rat and mouse.

[0029] The screening and isolation of the gene can be suitably carriedout by homology screening and PCR method.

[0030] For the resultant cDNA, it is possible to determine the basesequence by the conventional method, analyze the translation region anddetermine the amino acid sequence of the protein encoded by this, i.e.,URAT1.

[0031] It can be verified, for example, by the following method that theobtained gene is cDNA of the urate transporter gene, i.e., a geneproduct encoded by the cDNA is the urate transporter. The ability totransport (uptake) uric acid into cells can be confirmed by introducingcRNA (complementary RNA) prepared from the obtained URAT1 cDNA intooocyte to express, and measuring the uptake of a substrate into thecells by the conventional uptake experiment using uric acid as thesubstrate (Sekine, T. et al., Biochem. Biophis. Res. Commun.,251:586-591, 1998).

[0032] Also, transport property and substrate specificity of URAT1 canbe examined by applying the similar uptake experiment to expressingcells.

[0033] Further, the property of URAT1, for example, the property thatURAT1 performs the transport with time dependency, substrate selectivityand pH dependency of URAT1 can be examined by applying the similaruptake experiment to the expressing cells.

[0034] Homologous genes and chromosomal genes derived from the differenttissues or different organisms can be isolated by screening appropriatecDNA libraries or genomic DNA libraries made from the different genesources using cDNA of the obtained URAT1 gene.

[0035] Also, the gene can be isolated from the cDNA library by theconventional PCR method using synthetic primers designed on the basis ofthe information of the disclosed base sequence of the gene of thepresent invention (the base sequence represented by SEQ ID NO:1 or apart thereof).

[0036] The DNA libraries such as cDNA library and genomic DNA librarycan be prepared by the methods described in, for example, “Sambrook, J.,Fritsh E. F., and Maniatis, T., “Molecular Cloning” (published by ColdSpring Harbor Laboratory Press in 1989)”. Or when there is acommercially available library, it may be used.

[0037] To obtain the structure of URAT1 gene on human genome, thegenomic DNA library is screened using the obtained URAT1 gene cDNA, andthe obtained clones are analyzed. Or the structure may be searched onthe basis of the disclosed information of the human genome analysisresults using a homology search program.

[0038] The urate transporter (URAT1) of the present invention can beproduced by gene recombination technology using cDNA which encodes theurate transporter. For example, it is possible to incorporate DNA (cDNA,etc.) which encodes the urate transporter in an appropriate expressionvector and introduce the resultant recombinant DNA into appropriate hostcells. Expression systems (host vector system) for producing thepolypeptide include the expression systems of bacteria, yeast, insectcells and mammalian cells. Among these, to obtain the functionalprotein, it is desirable to use the insect cells and the mammaliancells.

[0039] For example, when the polypeptide is expressed in the mammaliancells, an expression vector is constructed by inserting DNA whichencodes the urate transporter in the downstream of an appropriatepromoter (e.g., SV40 promoter, LTR promoter, elongation 1α promoter andthe like) in an appropriate expression vector (e.g., retroviral vector,papilloma virus vector, vaccinia virus vector, SV40 type vector and thelike). Next, the target polypeptide is produced by transformingappropriate animal cells with the obtained expression vector andculturing transformants in an appropriate medium. The mammalian cells asthe hosts include cell lines such as monkey COS-7 cells, Chinese hamsterCHO cells, human HeLa cells and primary culture cells derived from renaltissues, LLC-PK1 cells derived from swine kidney, OK cells derived fromopossum kidney, and proximal convoluted tubule S1, S2 and S3 cellsderived from mouse.

[0040] As the cDNA which encodes the urate transporter URAT1, it ispossible to use the cDNA having the base sequence shown in the sequence1, and further it is possible to design DNA corresponding to the aminoacid sequence and use the DNA which encodes the polypeptide withoutbeing limited to the above cDNA. In this case, 1 to 6 codons whichencodes one amino acid are known, and the codon used may be optionallyselected, but it is possible to design the sequence with high expressionby considering use frequency of codons in the host utilized for theexpression. The DNA with the designed sequence can be acquired bychemical synthesis of DNA, fragmentation and bind of the above cDNA,partial modification of the base sequence and the like. The artificialpartial modification and mutagenesis can be carried out by site specificmutagenesis methods (Mark, D. F. et al., Proc. Natl. Acad. Sci. USA,18:5662-5666, 1984) utilizing primers including syntheticoligonucleotides which encode the desired modification.

[0041] The nucleotides (oligonucleotides or polynucleotides) whichhybridize with the urate transporter gene of the present invention undera stringent condition can be used as probes to detect the uratetransporter gene, and further can be used, for example, as antisenseoligonucleotides, ribozymes and decoys to modulate the expression of theurate transporter. As such nucleotides, it is possible to use, forexample, the nucleotides typically comprising the partial sequence ofconsecutive 14 or more bases or the complementary sequence thereof inthe base sequence represented by SEQ ID NO:1. In order to make thehybridization more specific, as the partial sequence, the longersequence, e.g., the sequence of 20 or more bases or 30 or more bases maybe used.

[0042] Also, using the urate transporter of the present invention or thepolypeptide having immunological equivalence thereto, it is possible toacquire antibodies thereof, and the antibodies can be utilized for thedetection and the purification of the urate transporter. The antibodycan be produced by using the urate transporter of the invention, afragment thereof, or a synthetic peptide having the partial sequencethereof and the like as an antigen. The polyclonal antibody can beproduced by the conventional method in which the antigen is inoculatedto the host animal (e.g., rat or rabbit) and immunized serum iscollected, and the monoclonal antibody can be produced by theconventional technology such as a hybridoma method.

[0043] Furthermore, the present invention provides a screening method ofa substance having uricosuric accelerating action. The protein of theinvention works for transporting uric acid into the cells and is deeplyinvolved in the reabsorption of uric acid. Also, as is shown in FIGS. 6,8, 9 and 10, it is possible to quantify the accelerating or inhibitingaction for uric acid uptake of the screening substance in the systemwhere the protein of the invention is expressed, by adding uric acid tothe system, further adding the screening substance thereto, andcomparing a uric acid uptake amount with that in the case with noaddition of the screening substance. As is shown in FIGS. 6 and 8, thesubstances clinically used as uricosuric accelerators have remarkablyinhibited the uptake of uric acid in the above experimental system, andthus, it is shown that it become possible to screen the uricosuricaccelerating action of the screening substance in this system. As thecells used in this screening system, the cells are not limited tooocytes used in the following experiments, and it is possible to usevarious living cells as long as the cells can express the protein of theinvention.

[0044] Therefore, the present invention provides the method forscreening substances having uricosuric regulating action using theprotein of the invention. As the uricosuric regulating actions, thereare the uricosuric accelerating action and the uricosuric inhibitingaction, and those having the uricosuric accelerating action arepreferable for the treatment/prevention of hyperuricemia and gout. Thus,the preferable uricosuric regulating action includes the uricosuricaccelerating action. Moreover, the present invention provides uricosuricregulators screened by the above screening method. The preferable uricacid regulator includes a uricosuric accelerators. The uricosuricregulator screened by the method of the invention can regulate theuptake of uric acid by the urate transporter involved in the uratetransport in the kidney, and therefore can be used as an activeingredient of the medicines for the treatment/prevention of variousdiseases associated with the reabsorption of uric acid such ashyperuricemia and gout.

[0045] It is possible to make the obtained active ingredient apharmaceutical composition using a pharmacologically acceptable carrier.

EXAMPLES

[0046] The present invention is described in more detail by examplesbelow, but these examples do not limit the invention.

[0047] In the following examples, unless otherwise specified, respectivemanipulations were carried out by the methods described in “Sambrook,J., Fritsch E. F., and Maniatis, T., “Molecular Cloning” (published byCold Spring Harbor Laboratory Press in 1989)” or when using commerciallyavailable kits, they were used according to the instructions of thecommercially available articles.

Example 1 Isolation of Kidney-Specific Urate Transporter (URAT1) cDNAand Analysis Thereof

[0048] On the basis of the base sequence information of OAT1, OAT2, OAT3and OAT4 already isolated by the present inventors, the disclosedanalysis results of the human genome project were searched using thehomology search program. As a result, multiple novel gene fragmentshaving homology to OAT1, OAT2, OAT3 and OAT4 were obtained. Among them,one of the novel gene fragments extremely close to the locus position ofOAT4 was analyzed, and the site thought to be the initiation codon wasidentified in it. This initiation codon was identified by comparing thenovel gene fragments with gene sequences of OAT1 and OAT4.

[0049] A primer specific for the 5′ upstream region of the predictedinitiation codon was made using 28 bases, and the isolation of thisnovel gene was attempted by 3′-RACE (3′-rapid amplification of cDNAends) method using messenger RNA derived from various tissues of human.As a result, a monoclone (URAT1) was obtained by the 3′-RACE methodusing human kidney messenger RNA. A single band obtained by PCR methodwas subcloned in pCRII-TOPO vector using TA cloning method, and furthersubcloned in pcDNA 3.1(+) vector which was the expression vector. As aresult, a novel cDNA (URAT1 cDNA) which has urate transport activity wasobtained (for analysis of transport function, see the followings.).

[0050] Determination of the base sequence of the c DNA (URAT1 cDNA)obtained by the above was carried out using specific primers by anautomatic sequencer (manufactured by Applied Biosystems) (described inSEQ ID NO:1).

[0051] The expression of URAT1 gene was analyzed in various tissues ofhuman (Northern blotting) (FIG. 1). Full length URAT1 cDNA was labeledwith ³²P-dCTP, and using this as a probe, hybridization was carried outusing filters (manufactured by Clontech) blotting RNA extracted fromvarious human tissues. The hybridization was carried out overnight in ahybridization solution comprising the labeled full length URAT1 cDNA,and the filters were washed with 0.1×SSC comprising 0.1% SDS at 65° C.As a result of Northern blotting, an intensive band was detected in therenal tissue. In human embryonic tissues, the band was detected in thekidney.

Example 2 Analysis of Urate Transporter Functions

[0052] From plasmid comprising URAT1 cDNA, cRNA (RNA complementary tocDNA) was prepared in vitro using T7 RNA polymerase (see Sekine, T., etal., J. Biol. Chem., 272:18526-18529, 1997).

[0053] The resultant cRNA was injected in oocytes of platanna, anduptake experiments of the radiolabeled uric acid in these oocytes werecarried out according to the method already reported (Sekine, T., etal., J. Biol. Chem., 272:18526-18529, 1997). As a result, it was foundthat the oocytes in which URAT1 was expressed showed uptake of [¹⁴C]uric acid as shown in FIG. 2. The oocytes in which URAT1 was expressedshowed time dependency in the uptake of [¹⁴C] uric acid. This indicatedthat not only URAT1 was bound to uric acid but also was the transporterto transport it into the cells. No uptake of [¹⁴C] PAH (para-aminohippuric acid) and [¹⁴C] TEA (tetraethylammonium) which are arepresentative substrate of the organic ion transporter family wasobserved (not shown).

[0054] Michaelis-Menten dynamic experiment in urate transport by URAT1was carried out. Concentration dependency of uric acid in the transportby URAT1 was studied by examining change of uptake amounts of uric acidat various concentrations by URAT1. The uptake experiment of theradiolabeled uric acid was carried out using oocytes injected with URAT1cRNA according to the method described above. As a result (FIG. 3), Kmvalue (Michaelis constant number) of the uric acid uptake wasapproximately 372±25 μM.

[0055] The effect of various electrolytes on the urate transport byURAT1 was studied (FIG. 4). When extracellular sodium was replaced withlithium, choline and N-methyl-D-glucamine (NMDG), the urate transportvia URAT1 was not changed. It was demonstrated that URAT1 was theextracellular sodium-independent urate transporter. When extracellularpotassium ions were completely replaced with sodium (0-K+ in FIG. 4) andsodium was completely replaced with the potassium ions (96 mM KCl), theurate transport was not also changed, which was demonstrated that URAT1was cell membrane potential-independent. When extracellular chlorideions were replaced with gluconic acid, the uptake of uric acid wassignificantly increased. From the experimental system using the isolatedcell membrane vesicle system, the presence of the exchanger for uricacid and chloride was shown at the side of renal tubular lumen in humankidney. Thus, this experimental result suggests that chloride might bethe exchange substrate of uric acid.

[0056] The pH dependency in the urate transport by URAT1 was studied. Asshown in FIG. 5, when the extracellular pH was acidified, the uratetransport in the oocyte injected with URAT1 cRNA was increased, but thisseems to be caused by non-specific absorption of uric acid in theoocytes injected with water (control). The substantial urate transport(URAT1-control) was not changed depending on pH.

Example 3 Study on Exchange Substrate of Uric Acid in the UrateTransporter

[0057] From the experimental system using the isolated cell membranevesicle system, it has been suggested that monocarboxylic acids such aslactic acid and nicotinic acid can be the exchange substrate of uricacid in the uric acid/anion exchanger in the human kidney. In order tostudy the exchange substrate of uric acid in URAT1, the oocytes werepreincubated with these monocarboxylic acids (1 mM), para-amino hippuricacid and ketoglutaric acid, and subsequently the transport of uric acidwas measured (FIG. 6). When the oocytes were preincubated with 1 mM ofpyrazine carboxylic acid and nicotinic acid (3-pyridine carboxylicacid), the uptake of uric acid was significantly increased in theoocytes injected with URAT1 cRNA. On the other hand, when the oocyteswere preincubated with para-amino hippuric acid and ketoglutaric acidwhich were not monocarboxylic acids, the uptake of uric acid was notfacilitated. The above results indicate that monocarboxylic acids suchas pyrazine carboxylic acid and nicotinic acid are the exchangesubstrate of uric acid.

[0058] In FIG. 6, when the oocytes were preincubated with lactic acidwhich was monocarboxylic acid, the uptake of uric acid was notfacilitated. It was thought to be occurred because the incorporatedlactic acid was transported outside of the cells via a pathway otherthan URAT1 due to abundant expression of endogenous lactate transportersin the oocytes. Also, it was anticipated that low affinity of lacticacid to URAT1 as shown below was also one of the causes. Therefore, 100nl of 100 mM non-radiolabeled L-lactic acid was precedently injected inthe oocytes, and then the uptake of the radiolabeled uric acid wasobserved (FIG. 7). When lactic acid was precedently injected, thesignificantly high uptake of uric acid was observed compared to the casewhere water was injected. Even when para-amino hippuric acid andketoglutaric acid were injected, no change was observed compared to thecase where water was injected (not shown).

[0059] From the results in FIGS. 6 and 7, URAT1 is the exchanger of uricacid and monocarboxylic acid. Pyrazinamide which is an antituberculousdrug is metabolized to become pyrazine carboxylic acid, which is thenexcreted into urine, whereas it is said to facilitate the reabsorptionof uric acid. The above result shows that as a result of the exchange ofuric acid and pyrazine carboxylic acid in URAT1, the uptake of uric acidis facilitated. Accordingly the mechanism to cause hyperuricemia hasbeen demonstrated which is a side effect of pyrazinamide which is theantituberculous drug.

Example 4 Screening of Inhibitory Substance for Urate Transporter

[0060] In order to further study substrate selectivity of URAT1, in theuptake experiment system of [¹⁴C] uric acid by the oocytes injected withURAT1 cRNA, various substances were added to the system and theireffects were examined (inhibitory experiments). The uptake experiment of[¹⁴C] uric acid was carried out using the oocytes injected with URAT1cRNA according to the method described above (FIGS. 8, 9 and 10). Theuptake of 50 μM [¹⁴C] uric acid was measured under the condition at pH7.4 in the presence and absence of various compounds (unlabeled) at theconcentrations shown in FIG. 8. As a result, various monocarboxylicacids (L-lactic acid, D-lactic acid, nicotinic acid, pyrazine carboxylicacid) significantly inhibited the transport of [¹⁴C] uric acid by URAT1(FIG. 8). Ketoglutaric acid which was dicarboxylic acid and could be theexchange substrate of OAT1 did not inhibit under the condition at pH7.4. Pyrazine dicarboxylic acid which had a similar structure topyrazine carboxylic acid showed slightly weak inhibitory effect. Anionicand cationic substances such as para-amino hippuric acid andtetraethylammonium did not show any inhibitory action (FIG. 8).

[0061] Medicines used for the treatment of hyperuricemia, such asprobenecid, benz-bromarone, sulfinpyrazon and phenylbutazone,significantly inhibited the uptake of uric acid in URAT1. Losartan whichis a drug for the treatment of hypertension and

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cgc cag cca cag tgg cag ctc ttg gac ccc aat gcc acg gcc462 Arg Phe Arg Gln Pro Gln Trp Gln Leu Leu Asp Pro Asn Ala Thr Ala 9095 100 105 acc agc tgg agc gag gcc gac acg gag ccg tgt gtg gat ggc tgggtc 510 Thr Ser Trp Ser Glu Ala Asp Thr Glu Pro Cys Val Asp Gly Trp Val110 115 120 tat gac cgc agc atc ttc acc tcc aca atc gtg gcc aag tgg aacctc 558 Tyr Asp Arg Ser Ile Phe Thr Ser Thr Ile Val Ala Lys Trp Asn Leu125 130 135 gtg tgt gac tct cac gct ctg aag ccc atg gcc cag tcc atc tacctg 606 Val Cys Asp Ser His Ala Leu Lys Pro Met Ala Gln Ser Ile Tyr Leu140 145 150 gct ggg att ctg gtg gga gct gct gcg tgc ggc cct gcc tca gacagg 654 Ala Gly Ile Leu Val Gly Ala Ala Ala Cys Gly Pro Ala Ser Asp Arg155 160 165 ttt ggg cgc agg ctg gtg cta acc tgg agc tac ctt cag atg gctgtg 702 Phe Gly Arg Arg Leu Val Leu Thr Trp Ser Tyr Leu Gln Met Ala Val170 175 180 185 atg ggt acg gca gct gcc ttc gcc cct gcc ttc ccc gtg tactgc ctg 750 Met Gly Thr Ala Ala Ala Phe Ala Pro Ala Phe Pro Val Tyr CysLeu 190 195 200 ttc cgc ttc ctg ttg gcc ttt gcc gtg gca ggc gtc atg atgaac acg 798 Phe Arg Phe Leu Leu Ala Phe Ala Val Ala Gly Val Met Met AsnThr 205 210 215 ggc act ctc ctg atg gag tgg acg gcg gca cgg gcc cga cccttg gtg 846 Gly Thr Leu Leu Met Glu Trp Thr Ala Ala Arg Ala Arg Pro LeuVal 220 225 230 atg acc ttg aac tct ctg ggc ttc agc ttc ggc cat ggc ctgaca gct 894 Met Thr Leu Asn Ser Leu Gly Phe Ser Phe Gly His Gly Leu ThrAla 235 240 245 gca gtg gcc tac ggt gtg cgg gac tgg aca ctg ctg cag ctggtg gtc 942 Ala Val Ala Tyr Gly Val Arg Asp Trp Thr Leu Leu Gln Leu ValVal 250 255 260 265 tcg gtc ccc ttc ttc ctc tgc ttt ttg tac tcc tgg tggctg gca gag 990 Ser Val Pro Phe Phe Leu Cys Phe Leu Tyr Ser Trp Trp LeuAla Glu 270 275 280 tcg gca cga tgg ctc ctc acc aca ggc agg ctg gat tggggc ctg cag 1038 Ser Ala Arg Trp Leu Leu Thr Thr Gly Arg Leu Asp Trp GlyLeu Gln 285 290 295 gag ctg tgg agg gtg gct gcc atc aac gga aag ggg gcagtg cag gac 1086 Glu Leu Trp Arg Val Ala Ala Ile Asn Gly Lys Gly Ala ValGln Asp 300 305 310 acc ctg acc cct gag gtc ttg ctt tca gcc atg cgg gaggag ctg agc 1134 Thr Leu Thr Pro Glu Val Leu Leu Ser Ala Met Arg Glu GluLeu Ser 315 320 325 atg ggc cag cct cct gcc agc ctg ggc acc ctg ctc cgcatg ccc gga 1182 Met Gly Gln Pro Pro Ala Ser Leu Gly Thr Leu Leu Arg MetPro Gly 330 335 340 345 ctg cgc ttc cgg acc tgt atc tcc acg ttg tgc tggttc gcc ttt ggc 1230 Leu Arg Phe Arg Thr Cys Ile Ser Thr Leu Cys Trp PheAla Phe Gly 350 355 360 ttc acc ttc ttc ggc ctg gcc ctg gac ctg cag gccctg ggc agc aac 1278 Phe Thr Phe Phe Gly Leu Ala Leu Asp Leu Gln Ala LeuGly Ser Asn 365 370 375 atc ttc ctg ctc caa atg ttc att ggt gtc gtg gacatc cca gcc aag 1326 Ile Phe Leu Leu Gln Met Phe Ile Gly Val Val Asp IlePro Ala Lys 380 385 390 atg ggc gcc ctg ctg ctg ctg agc cac ctg ggc cgccgc ccc acg ctg 1374 Met Gly Ala Leu Leu Leu Leu Ser His Leu Gly Arg ArgPro Thr Leu 395 400 405 gcc gca tcc ctg ttg ctg gcg ggg ctc tgc att ctggcc aac acg ctg 1422 Ala Ala Ser Leu Leu Leu Ala Gly Leu Cys Ile Leu AlaAsn Thr Leu 410 415 420 425 gtg ccc cac gaa atg ggg gct ctg cgc tca gccttg gcc gtg ctg ggg 1470 Val Pro His Glu Met Gly Ala Leu Arg Ser Ala LeuAla Val Leu Gly 430 435 440 ctg ggc ggg gtg ggg gct gcc ttc acc tgc atcacc atc tac agc agc 1518 Leu Gly Gly Val Gly Ala Ala Phe Thr Cys Ile ThrIle Tyr Ser Ser 445 450 455 gag ctc ttc ccc act gtg ctc agg atg acg gcagtg ggc ttg ggc cag 1566 Glu Leu Phe Pro Thr Val Leu Arg Met Thr Ala ValGly Leu Gly Gln 460 465 470 atg gca gcc cgt gga gga gcc atc ctg ggg cctctg gtc cgg ctg ctg 1614 Met Ala Ala Arg Gly Gly Ala Ile Leu Gly Pro LeuVal Arg Leu Leu 475 480 485 ggt gtc cat ggc ccc tgg ctg ccc ttg ctg gtgtat ggg acg gtg cca 1662 Gly Val His Gly Pro Trp Leu Pro Leu Leu Val TyrGly Thr Val Pro 490 495 500 505 gtg ctg agt ggc ctg gcc gca ctg ctt ctgccc gag acc cag agc ttg 1710 Val Leu Ser Gly Leu Ala Ala Leu Leu Leu ProGlu Thr Gln Ser Leu 510 515 520 ccg ctg ccc gac acc atc caa gat gtg cagaac cag gca gta aag aag 1758 Pro Leu Pro Asp Thr Ile Gln Asp Val Gln AsnGln Ala Val Lys Lys 525 530 535 gca aca cat ggc acg ctg ggg aac tct gtccta aaa tcc aca cag ttt 1806 Ala Thr His Gly Thr Leu Gly Asn Ser Val LeuLys Ser Thr Gln Phe 540 545 550 tagcctcctg aggaacctgc gatgggacggtcagaggaag agacttcttc tgttctctgg 1866 agaaggcagg aggaaagcaa agacctccatttccagaggc ccagaggctg ccctctgagg 1926 tccccactct cccccagggc tgcccctccaggtgagccct gcccctctca cagtccaagg 1986 ggcccccttc aatactgaag gggaaaaggacagtttgatt ggcaggaggt gacccagtgc 2046 accatcaccc tgccctgccc tcgtggcttcggagagcaga ggggtcaggc ccaggggaac 2106 gagctggcct tgccaaccct ctgcttgactccgcactgcc acttgtcccc ccacacccgt 2166 ccacctgccc agagctcaga gctaaccaccatccatggtc aagacctctc ctagctccac 2226 acaagcagta gagtctcagc tccacagctttacccagaag ccctgtaagc ctggcccctg 2286 gcccctcccc atgtccctcc aggcctcagccacctgcccg ccacatcctc tgcctgctgt 2346 ccccttccca ccctcatccc tgaccgactccacttaaccc ccaaacccag ccccccttcc 2406 aggggtccag ggccagcctg agatgcccgtgaaactccta cccacagtta cagccacaag 2466 cctgcctcct cccaccctgc cagcctatgagttcccagag ggttggggca gtcccatgac 2526 cccatgtccc agctccccac acagcgctgggccagagagg cattggtgcg agggattgaa 2586 taaagaaaca aatgaaaaaa aaaaaaaaaaaaaaaaaaaa aaaaaaaaaa aaaaaa 2642 2 553 PRT Homo sapiens 2 Met Ala PheSer Glu Leu Leu Asp Leu Val Gly Gly Leu Gly Arg Phe 1 5 10 15 Gln ValLeu Gln Thr Met Ala Leu Met Val Ser Ile Met Trp Leu Cys 20 25 30 Thr GlnSer Met Leu Glu Asn Phe Ser Ala Ala Val Pro Ser His Arg 35 40 45 Cys TrpAla Pro Leu Leu Asp Asn Ser Thr Ala Gln Ala Ser Ile Leu 50 55 60 Gly SerLeu Ser Pro Glu Ala Leu Leu Ala Ile Ser Ile Pro Pro Gly 65 70 75 80 ProAsn Gln Arg Pro His Gln Cys Arg Arg Phe Arg Gln Pro Gln Trp 85 90 95 GlnLeu Leu Asp Pro Asn Ala Thr Ala Thr Ser Trp Ser Glu Ala Asp 100 105 110Thr Glu Pro Cys Val Asp Gly Trp Val Tyr Asp Arg Ser Ile Phe Thr 115 120125 Ser Thr Ile Val Ala Lys Trp Asn Leu Val Cys Asp Ser His Ala Leu 130135 140 Lys Pro Met Ala Gln Ser Ile Tyr Leu Ala Gly Ile Leu Val Gly Ala145 150 155 160 Ala Ala Cys Gly Pro Ala Ser Asp Arg Phe Gly Arg Arg LeuVal Leu 165 170 175 Thr Trp Ser Tyr Leu Gln Met Ala Val Met Gly Thr AlaAla Ala Phe 180 185 190 Ala Pro Ala Phe Pro Val Tyr Cys Leu Phe Arg PheLeu Leu Ala Phe 195 200 205 Ala Val Ala Gly Val Met Met Asn Thr Gly ThrLeu Leu Met Glu Trp 210 215 220 Thr Ala Ala Arg Ala Arg Pro Leu Val MetThr Leu Asn Ser Leu Gly 225 230 235 240 Phe Ser Phe Gly His Gly Leu ThrAla Ala Val Ala Tyr Gly Val Arg 245 250 255 Asp Trp Thr Leu Leu Gln LeuVal Val Ser Val Pro Phe Phe Leu Cys 260 265 270 Phe Leu Tyr Ser Trp TrpLeu Ala Glu Ser Ala Arg Trp Leu Leu Thr 275 280 285 Thr Gly Arg Leu AspTrp Gly Leu Gln Glu Leu Trp Arg Val Ala Ala 290 295 300 Ile Asn Gly LysGly Ala Val Gln Asp Thr Leu Thr Pro Glu Val Leu 305 310 315 320 Leu SerAla Met Arg Glu Glu Leu Ser Met Gly Gln Pro Pro Ala Ser 325 330 335 LeuGly Thr Leu Leu Arg Met Pro Gly Leu Arg Phe Arg Thr Cys Ile 340 345 350Ser Thr Leu Cys Trp Phe Ala Phe Gly Phe Thr Phe Phe Gly Leu Ala 355 360365 Leu Asp Leu Gln Ala Leu Gly Ser Asn Ile Phe Leu Leu Gln Met Phe 370375 380 Ile Gly Val Val Asp Ile Pro Ala Lys Met Gly Ala Leu Leu Leu Leu385 390 395 400 Ser His Leu Gly Arg Arg Pro Thr Leu Ala Ala Ser Leu LeuLeu Ala 405 410 415 Gly Leu Cys Ile Leu Ala Asn Thr Leu Val Pro His GluMet Gly Ala 420 425 430 Leu Arg Ser Ala Leu Ala Val Leu Gly Leu Gly GlyVal Gly Ala Ala 435 440 445 Phe Thr Cys Ile Thr Ile Tyr Ser Ser Glu LeuPhe Pro Thr Val Leu 450 455 460 Arg Met Thr Ala Val Gly Leu Gly Gln MetAla Ala Arg Gly Gly Ala 465 470 475 480 Ile Leu Gly Pro Leu Val Arg LeuLeu Gly Val His Gly Pro Trp Leu 485 490 495 Pro Leu Leu Val Tyr Gly ThrVal Pro Val Leu Ser Gly Leu Ala Ala 500 505 510 Leu Leu Leu Pro Glu ThrGln Ser Leu Pro Leu Pro Asp Thr Ile Gln 515 520 525 Asp Val Gln Asn GlnAla Val Lys Lys Ala Thr His Gly Thr Leu Gly 530 535 540 Asn Ser Val LeuLys Ser Thr Gln Phe 545 550

1. A protein comprising an amino acid sequence represented by SEQ ID NO:1 or an amino acid sequence derived therefrom by deletion, substitution or addition of one to several amino acids and being capable of transporting uric acid and its analogs.
 2. The protein according to claim 1 which is derived from human.
 3. The protein according to claim 1 which is derived from organs, tissues or cultured cells.
 4. A gene encoding the protein according to claim
 1. 5. DNA comprising the base sequence represented by SEQ ID NO:1 or DNA hybridizing with the DNA under a stringent condition and encoding a protein being capable of transporting uric acid and its analogs or exchanging uric acid and the other anion.
 6. The gene according to claim 5, which is derived from human.
 7. The gene according to claim 5, which is derived from organs, tissues or cultured cells.
 8. The gene according to claim 4 or a plasmid comprising a gene encoding a protein of the gene.
 9. The plasmid according to claim 8, wherein the plasmid is an expression plasmid.
 10. Host cells transformed with the plasmid according to claim
 8. 11. Nucleotides comprising a partial sequence of consecutive 14 bases or more in the base sequence represented by SEQ ID NO:1 or a complementary sequence thereof.
 12. The nucleotides according to claim 11, used as a probe to detect the gene encoding the protein being capable of transporting uric acid and its analogs or exchanging uric acid and other anion.
 13. The nucleotides according to claim 11, used to modulate the expression of the gene encoding the protein being capable of transporting uric acid and its analogs or exchanging uric acid and other anion.
 14. An antibody against the protein according to claim
 1. 15. A method for detecting an action of a subject substance as a substrate for an ability to transport uric acid of the protein and its analogs or exchange uric acid and other anion using the protein according to claim
 1. 16. A method for screening substances having a uricosuric regulating action using the protein according to claim
 1. 17. A uricosuric regulating agent capable of being screened by the method according to claim
 16. 18. A method for changing kinetics of uric acid and its analogs transported by the protein in the kidney, by modulating an ability to transport uric acid of the protein and its analogs or exchange the uric acid and other anion, using the protein according to claim 1, a specific antibody thereof, a function accelerating substance thereof or a function inhibiting substance thereof.
 19. A method for changing effects of uric acid and its analogs transported by the protein on kinetics in the kidney, by modulating an ability to transport uric acid of the protein and its analogs or exchange the uric acid and other anion, using the protein according to claim 1, a specific antibody thereof, a function accelerating substance thereof or a function inhibiting substance thereof.
 20. A method for changing effects of uric acid and its analogs transported by the protein on total blood concentrations, by modulating an ability to transport uric acid of the protein and its analogs or exchange the uric acid and other anions, using the protein according to claim 1, a specific antibody thereof, a function accelerating substance thereof or a function inhibiting substance thereof.
 21. A method for detecting and changing effects of uric acid and its analogs transported by the protein on kinetics in the kidney, by excessively expressing the protein in the certain cells or by modulating an ability to transport uric acid of the protein existing in the cells and its analogs, using the protein according to claim 1, a specific antibody thereof, a function accelerating substance thereof or a function inhibiting substance thereof, and cDNA (complementary DNA) encoding the same. 